Antimitotic compounds

ABSTRACT

Antimitotic terpenoid compounds including sarcodictyin A and as eleutherobin may be obtained from organisms of the order Gorgonacea. Methods of preparing such compounds provided, as are novel antimitotic diterpene compounds having formula (I).

BACKGROUND OF THE INVENTION

Antimitotic compounds interfere with the dynamiic assembly anddisassembly, of α-and β-tubulin into microtubules causing cells toarrest in mitosis. Prolonged arrest in mitosis eventually leads to celldeath, often by apoptosis. Two chemical classes of antimitotic agents,the vinca alkaloids (vinblastine, vincristine, and vinorelbine) and thetaxanes (paclitaxel and docetaxel), are clinically useful anticancerdrugs. Most known antinmitotic agents induce mitotic arrest byinhibiting the polymerization of tubulin into microtubules. This is themechanism of the vinca alkaloids and rhizoxin.

Paclitaxel was the first chemical entity shown to cause mitotic arrestby stabilzing microtubules against depolymerization. Four additionalchemotypes that have paclitaxel-like effects were later identified.These include the myxobacterium metabolites epothilones A and B, themarine sponge metabolites discodermolide, laulimalide, andisolaulimalide, and the soft coral terpenoid, eleutherobin (shown belowas Compound 1.) Ojirna et al. (1999) Proc. Natl. Acad. Sci. USA96:4256-4261, propose a common pharmacophore for the microtubulestabilizing compounds that effectively accommodates nonataxel,paclitaxel, discodermolide, eleutherobin, and the epothilones. Thismodel predicts that three regions of eleutherobin (boxes A, B, and Cbelow) are important for binding to tubulin (Me=methyl; Ac=acetyl).

The majority of known antimitotic natural products were initiallyisolated because they exhibited potent in vitro cytotoxicity. Onlysubsequent detailed mechanism of action studies revealed that theyarrested cells in mitosis and interfered with tubulin assembly anddisassembly dynamics. For example, rhizoxin is a 16-membered ringmacrolide first isolated in 1984 and determined to be very cytotoxic.Only later was rhizoxin shown to cause the accumulation of cells inmitosis. Sarcodictyins A-D were the first members of a cytotoxicterpenoid class of compounds to be identified (see: D'Ambrosio, M., etal. (1987) Helv. Chim. Acta. 70:2019-2027; and, (1988) Helv. Chim. Acta.71:964-976), their paclitaxel-like properties being recognized onlylater.

Eleutherobin, a diterpene glycoside, was originally isolated from thesoft coral Eleutherobia sp. (possibly E. albiflora) collected in WesternAustralia (see: Lindel, T. et al. (1997) J. Am. Chem. Soc.119:8744-8745; and, international patent application published May 23,1996 under WO 96/14745). Subsequently, eleuthosides A and B wereisolated from a different species of Eleutherobia (E. aurea). Theeleuthosides differ from eleutherobin by the presence of a hydroxylsubstitute at the C-4 position shown above (rather than a methoxylsubstitute) and, as shown above, by the presence of an acetyl group atthe 3″ or the 4″ position of the arabinose moiety shown above, inaddition to an acetyl at the 2″ position (Ketzinel, S., et al. (1996) J.Nat. Prod. 59:873-875). Later, a total synthesis of eleutherobin andeleuthosides A and B was reported (Nicolaou, K. C., et al. (1998) J. Am.Chem. Soc. 120:8674-8680). As reported in the latter reference, theeleuthosides may be made by converting C-4 ketal precursors to C-4hydroxyl forms.

SUMMARY OF THE INVENTION

Using a new cell-based antimitotic assay, the inventors herein havedemonstrated potent antimitotic activity in extracts of marine organismsproviding abundant new sources of antimitotic terpenoids. Microscopicexamination of cells arrested in mitosis by the extracts show tubulinbundling, similar to the effects of paclitaxel. Bioassay guidedfractionation of extracts of marine organisms has led to the isolationof eleutherobin 1 and the novel diterpenes shown below, includingdesmethyleleutherobin 2, desacetyleleutherobin 3, isoeleutherobin A 4,Z-cleutherobin 5, carnbaeoside 6, and caribaeolin 7.

This invention provides the use of organisms of the order Gorgonacea asa source for the preparation of purified or partially purifiedantimitotic compounds, including terpenoids. This invention provides amethod to obtain antimitotic terpenoids wherein an extract of anorganism of the order Gorgonacea in a solvent is subjected tofractionation to separate antimitotic compounds from compounds lackingantimitotic activity. Fractionation may include any suitable process forseparation of terpenoid compounds. Antimitotic terpenoids may compriseone or more of the compounds identified as Compounds 1-7 above as wellas sarcodictyin A. Preferably the organism employed is a gorgonian coralsuch as Erythropodium caribaeorum.

A solvent used for preparing extracts of organisms according to thisinvention may be any suitable solvent for extraction or dissolution ofterpenoids, including alcohols (e.g. methanol, ethanol), acetone,acetate compounds, chloroform, dichloromethane, etc. Mixtures of polarsolvents with water may be used, the ratios to be determined byprocedures known in the art. In some applications, it will be preferredthat the solvent be one that is incapable of forming a ketal compound,which excludes the alcohols. A particularly preferred solvent is anacetate such as ethyl acetate (EtOAc).

Preferred fractionation procedures are chromatographic. Preferably,several chromatography procedures will be performed, with each procedureintended to separate compounds according to differing parameters suchas: solubility (e.g. gradient elution), and molecular size (e.g. by useof a molecular sieve such as a Sephadex™ gel). A suitable gradientelution chromatography procedure involves elution of compounds from asubstrate (e.g. a silica bed in a column) by application of mixedsolvents having varying ratios of solvent components (e.g. reversed ornormal phase; vacuum or flash liquid chromatography). For example,applied solvents may have varying ratios of a polar solvent (e.g.methanol: MeOH) to either: a different polar solvent (e.g. EtOAc orH₂O), or a non-polar solvent (e.g. hexane). Selection of appropriate bedsubstrates and elution profiles as well as chromatography bed design maybe done using standard laboratory procedures and protocols, or thespecific procedures described herein may be employed. Purification mayalso be accomplished by using high pressure liquid chromatography (HPLC)which may be used to particular advantage as a final step inpurification. In some cases, purification by crystallization ofcompounds from solution may be accomplished.

Fractionation of compounds in this invention may be guided by monitoringfor particular chemical or physical characteristics of desired orundesired compounds. Monitoring for the specific characteristics of suchcompounds as described herein may be carried out using standardprocedures, such as determination of melting/decomposition temperatureor by spectroscopic methods (including mass spectrometry, UVspectrometry and nuclear magnetic resonance (NMR)). For example, theunique UV chromophore of eleutherobin may be used to monitor thepresence of that compound in fractions obtained as the method of thisinvention is carried out.

The method of this invention may also be guided by the use of anysuitable test or assay for anrimitotic activity. Presence or absence ofantimitotic compounds in crude extracts of selected organisms of theabove-mentioned orders may be determined prior to the performance of themethod of this invention. Further, such an assay may be used to monitorthe presence of desired compounds in fractions obtained duringperformance of the method of this invention. Microscopic examination ofcells treated with a test substance is a traditional test forantimitotic activity. Other suitable assays are disclosed herein.

This invention also provides an assay for antimitotic activitycomprising:

(a) applying a sample to be tested for antimitotic activity to cellswhich are capable of mitosis in culture;

(b) culturing the cells for a time sufficient for the cells to undergomitosis;

(c) fixing the cells on a substrate and treating the cells to increasethe cells' permeability to an antibody; and

(d) applying a mitotic cell-specific antibody to the cells and detectingbinding of said antibody within the cells.

In the above-described assay, cells may be fixed using any suitablemethod for the type of cell and the substrate. Formaldehyde is a commonfixative. Permeability may be increased by treatment in known ways,including treatment with an alcohol and/or a detergent. A preferredmethod of detecting binding of the antibody is to apply a secondantibody capable of binding to the mitotic cell-specific antibody usedin (d). The second antibody is typically linked to a detectableindicator. After removal of unbound antibodies from the cells, thepresence of bound mitotic cell-specific antibody is detected bydetermining the presence of the detectable indicator. When thedetectable indicator is an enzyme, its presence is determined bydetermining the presence of a product of the reaction that is catalyzedby the enzyme.

This invention also provides an antimitotic compound and pharmaceuticalpreparations thereof, wherein the compound has the formula:

wherein Me is methyl; R₁, R₂ and R₃ are independently selected from thegroup consisting of H and a C₁-C₆ acyl; R₄ is selected from the groupconsisting of H₂Me and a substituted or unsubstituted straight-chain,branched, or cyclic C₁-C₁₀ alkyl; providing that if R₄ is Me, R₁ is notacyl; and, providing that if R₄ is H, two of R₁, R₂ and R₃ are not acyl.Preferably, the acyl is acetyl and the alkyl is a C₂-C₅ straight-chainor branched moiety. A compound of this invention includes salts(preferably pharmaceutically acceptable salts) and also includesisomers, including those of the Z and E configurations and those of theα and β configurations at the glycosidic bond.

Preferred embodiments of this invention include those in which: R₄ is H,ethyl, propyl, butyl or pentyl; one of R₁ and R₂ is H and the other Ac:and, R₃ is H. In another preferred embodiment: R₄ is Me; R₁ and R₃ areH; and, R₂ is Ac.

A compound of this invention may be isolated from natural sources asdescribed herein; prepared by total synthesis by adapting the methods ofNicolaou, K. C., et al. [supra]; or, from an intermediate. Theintermediate may be prepared by total synthesis using conventionalstarting materials or obtained by reduction and glycosylation ofsarcodictyin A (see: WO 96/14745). An intermediate used in thepreparation of compounds of this invention may be isoeleutherobin A,desmethyleleutherobin or eleutherobin, with appropriate substitutions atR₁₋₃ done using conventional procedures (such as the acetylationprocedure described in the Examples below or by Nicolaou, K. C., et al.[supra]) and substitutions at C-4 done according to the methodsdescribed by Nicolaou, K. C., et al. [supra].

This invention also provides a method of converting a diterpenoidcompound, including a compound of this invention having a hydroxylsubstituent at C-4 to a compound having a ketal substituent at C-4 bycontacting a compound having the hydroxyl substituent with an alcohol inthe presence of a suitable acid catalyst. The catalyst may be an acidbut is preferably a catalyst such as pyridinium p-toluenesulfonate. Thealcohol may be methanol or any substituted or unsubstituted,straight-chain, branched, or cyclic alcohol having from 2-10 carbonatoms. Preferred alcohols are methanol, ethanol, the propanols, thebutanols and the pentanols (to provide methoxyl, ethoxyl, propanoxyl,butanoxyl or pentanoxyl substituents respectively, at C-4.)

This invention also provides the use of a compound or a pharmaceuticalpreparation of this invention as an antimitotic agent and for thepreparation of antimilotic medicaments. This invention also provides amethod for causing mitotic arrest in one or more cells present in a cellpopulation, comprising treating the cell population with a sufficientamount of a compound or pharmaceutically acceptable salt thereof, apharmaceutical preparation or medicament of this invention to arrestmitosis in one or more cells in the cell population. The cell populationmay be a population of cancerous cells, including a tumor. This methodmay be performed in vitro or may be performed in vivo throughadministration to a human or animal patient with a cancer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 (A and B) are graphs showing mitotic arrest of MCF-7 cells bydifferent concentrations of paclitaxel, as determined by mitotic spreadsand microscopy (FIG. 1A) and the ELISA (□) and ELLCA(▪) assays describedherein (FIG. 1B).

FIG. 2 (A and B) are graphs showing mitotic arrest of MCF-7 cells usingthe indicated compounds at different concentrations, as deternined bythe ELICA essay described herein.

FIG. 3 (A and B) are panels in a schematic showing a fractionationprocedure according to exemplified embodiments of this invention. E.caribaeorum is homogenized to produce a crude extract. The crude extractis subjected to fractionation procedures including reversed and normalphase chromatography followed by HPLC, to produce antimitotic compounds.

FIG. 4 is a graph comparing mitotic arrest of MCF-7 cells by differentdoses of eleutherobin (∘) and desmethyleleutherobin () as determined bymicroscopic examination of the cells.

FIG. 5 is a graph showing the effect of different doses ofdesmethyleleutherobin on squamous cell carcinoma in mice.

DETAILED DESCRIPTION

Previously, the only known natural source of eleutherobin was a speciesof soft coral from Western Australia (see: Lindel, T. et al. [supra].This invention provides an abundant new source of antimitotic terpenoidsfrom a taxonomic order of coral and coral-like organisms much differentfrom the order Alcyoniidae which comprises the soft coral described byLindel, T. et al. Using assays specifically adapted to detectantimitotic compounds, it has now been determined that organisms of theorder Gorgonacea produce such antimitolic compounds. Such organismsinclude species of the genus Erythropodium; species of the genusRumphella (family Gorgoniidae); Mopsea whiteleggei and Muncellisis Sp. a(family Isididae); Subergorgia Sp. 1 cf Mollis and Subergorgia Mollis(geog. variant) (family Subergorgiidae); and, Junceella sp. d.Verrucella Sp. b and Ctenosella regia (family Ellisellidae).

According to this invention, a preferred source of antimitotic compoundsare the gorgonian corals, and in particular, Eryrhropodium caribaeorum.Gorgonian corals are found in all tropical and sub-tropical regions,particularly the Caribbean. These corals are found in abundance, andhave been grown in aquarium environments and may be readily identified(for example, see: Bayer, F. M.; “She Shallow-Water Octocorallia of theWest Indian Region” (1961) Martinus Nighoff; The Hague, at page 65 and75-77 for Erythropodium). E. caribaeorum may be collected in abundancefrom southern Florida to the Virgin Islands. An analysis of toxic ordefensive compounds of the latter species has been reported. Thisinvestigation included fractionation and HPLC analysis of some terpenoidcompounds but did not reveal the presence of the compounds of thisinvention, nor any compounds having the activity of the compounds asdescribed herein (Fenical, W. and Pawlik, J. R. (1991) March Ecol. Prog.Ser. 75:1-8).

Assays suitable for detection of antimitotic compounds may be based onthe use of antibodies specific for mitotic cells, such as thosedescribed in the international patent application published Apr. 1, 1999under WO 99/15157. Such an assay will typically employ cells whichregularly divide in culture (e.g. cancer cells). A known antimitoticcompound such as nocodazole may be used as a control. In the assay,determination of the cells which proceed to mitosis is carried out usingany of the known immunological methods by employing antibodies whichhave specificity for mitotic cells. Monoclonal antibodies demonstratingsuch specificity are known and include MPM-2 which was raised againstmitotic HeLa cells and recognizes phospho-epitopes that are highlyconserved in mitotic proteins of all eukaryotic species. Other examplesare the monoclonal antibodies recognizing phospho-epitopes in the pairedhelical filament proteins (PHF) found in brain tissue of patientssuffering from Alzheimer's disease as described in: PCT InternationalApplication published Jul. 4, 1996 under No. WO 96/20218; and, Vincentet al. (1996) “The Journal of Cell Biology”, 132:413-425. The examplesin this specification make use of the antibody TG-3 described in thelatter two references, which may be obtained from Albert EinsteinCollege of Medicine of Yeshiva University, Bronx, N.Y.

The TG-3 monoclonal antibody, originally described as a marker ofAlzheimer's disease, is highly specific for mitotic cells. Flowcytometry shows that TG-3 immunofluorescence is >50-fold more intense inmitotic cells than in interphase cells. In Western blots, the antibodyreacts with a 105-kDa protein identified as a mitotically phosphorylatedform of nucleolin, that is present in abundance in extracts of cellstreated for 20 hours with the antimitotic agent nocodazole but presentat only low levels in extracts from cycling MCF-7 cells. Densitometricscanning of the bands on Western blots in these examples show a 27-folddifference in intensity between nocodazole-treated and untreated cells,corresponding well to the difference in the number of mitotic cells inthe two samples: 80% for the nocodazole-treated sample and 3% for theuntreated sample, as measured by microscopy.

TG-3 also recognizes mitotic cells in EUISA. In the ELISA assay, thecells may be grown in multi-well plates, lysed and transferred toprotein-binding ELISA plates for adsorption to the plastic surface. Theantigen may be detected by incubating with TG-3 antibody, anHRP-conjugated secondary antibody and performing a calorimetricdetermination of HRP activity.

Immunological methods useful for determination of mitotic cells in anassay include any method for determining antibody-antigen binding,including: inimunocytochernistry (e.g. inmnunofluorescence), flowcytometry, immunoblotting, and ELISA. Several immunological methods aredescribed in detail in examples herein as well as in Vincent, I. et al.[supra]. Other immunological procedures not described herein are wellknown in the art and may be readily adapted for use in an assay employedin this invention. However, high throughput testing of samples may bestbe achieved by use of ELISA or the ELICA assay described herein.

Pharmaceutical preparations containing compounds of this invention maybe prepared as for similar preparations containing eleutherobin,paclitaxel, etc. In the case of compounds of this invention capable ofsalt formulation, pharmaceutically acceptable salts (e.g. HCI salt) maybe used to advantage to permit administration of the compound in anaqueous solvent. Modes of administration to an animal or human patientinclude intravenous and intraperitoneal, to achieve a circulatingconcentration of the drug as predicted from its activity using standardmethodology.

EXAMPLES

Sample Collection and Extract Preparation. Specimens of marineinvertebrates were collected/by hand, using scuba, from cold temperatewaters of the Pacific Ocean along the coast of British Columbia(Canada), from tropical Pacific Ocean reefs off Motupore and Madang inPapua New Guinea, and from tropical waters off the Island of Dominica inthe Caribbean. Samples were deep frozen on site and transported over dryice. Voucher samples of each invertebrate were stored in methanol at−20° C. at The University of British Columbia, Vancouver, B.C. Canada,for taxonomic identification. Marine microorganisms were isolated fromthe invertebrates on site using marine culture media, and pure cultureswere grown as a lawn on solid agar marine media in 10 cm petri platesfor several days and then freeze-dried.

Extracts of invertebrates were prepared by homogenizing in methanolapproximately 200 g of each sample. The homogenates were filtered andconcentrated to dryness in vacuo to give a gummy residue. Extracts ofmicroorganisms were prepared by extracting the freeze-dried culture(cells and agar) multiple times with dry methanol/acetone, followed bylyophilization. A small amount of each extract was dissolved in DMSO forthe antimitotic screening assay.

Cell Culture and Treatment. Human breast carcinoma MCF-7 cells werecultured as monolayers. The cells were seeded at 10,000 per well of96-well polystyrene tissue culture plates (Falcon) in 200 μl medium andwere allowed to grow overnight. Crude extracts of marine organisms werethen added at about 10 μg/ml or 1 μg/ml, from 1000-fold stocks indimethylsulfoxide (DMSO). Untreated samples received an equivalentamount of DMSO and several as negative controls. Cells treated with 100ng/ml nocodazole (Sigma), from a 1000-fold stock in DMSO, served aspositive controls. Cells were incubated for 16-20 hours. The relativenumber of cells in mitosis was then determined by microscopy, byenzyme-linked immunosorbent assay (ELISA) or by an enzyme-linkedcytochemical assay (ELICA), as described below.

ELISA of Mitotic Cells. After incubation with marine organism extracts,the cell culture medium was withdrawn carefully using a pipetor.Rounded-up mitotic cells remained attach to the plates. The cells werelysed by adding 100 μl of ice-cold lysis buffer (1 mM EGTA pH 7.4, 0.5mM phenylmethylsulfonyl fluoride) and by pipeting up-and-down ten times.The cell lysates were transferred to 96well PolySorp™ plates (Nunc) anddried completely in a stream of air at about 37° C. from a hair dryer.Vacant protein binding sites were blocked by adding a 200 μl of antibodybuffer (10 mM Tri-HCl pH 7.4, 150 mM NaCl, 0.1 mM phenylmethylsulfonylfluoride, 3% (w/v) dried nonfat milk (Carnation)) per well for 1 hour atroom temperature. This was removed and replaced with 100 μl antibodybuffer containing 0.1-0.5 μg/ml TG-3 monoclonal antibody. After 16-20hour incubation at 4° C., the antibody solution was removed and thewells were rinsed twice with 200 μl 10mM Tris-HCl pH 7.4, 0.02% Tween20™. Horseradish peroxidase (HRP) conjugated goat anti-mouse IgMsecondary antibody (Southern Biotechnology Associates) was added at a500-fold dilution. After overnight incubation at 4° C., the antibodysolution was removed and the wells were rinsed three times with 200 μl10 mM Tris-HCl pH 7.4, 0.02% Tween 20™. Finally, 100 μl of 120 mMNa₂HPO₄, 100 mM citric acid (pH 4.0) containing 0.5 μg/ml 2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) and 0.01% hydrogen peroxide wasadded for 1 hour at room temperature and absorbance at 405 nm wasdetermined using a Dynex MRX™ plate reader.

ELICA of Miltotic Cells. While the ELISA is accurate and reliable, itrequires transferring cell lysates to ELISA plates and many solutionchanges. The assay of this invention is faster and easier to use fordrug screening. This assay, combining some features of ELISA and the“cytoblot” technique (Stockwell, B. R. et al. (1999) Chemistry andBiology 6:71-93), reduces the time of the procedure and the number ofsteps by half and does not require transfer of samples to ELISA plates.In a preferred assay of this invention, cells are fixed withformaldehyde in their microtiter culture plates and permeabilized withmethanol and detergents. The TG-3 primary antibody and HRP-conjugatedsecondary antibody may be added sequentially but are preferably addedsimultaneously. Colorimetric detection of HRP activity remainsunchanged. This new assay is termed: an Enzyme-LinkedImmuno-Cytochemical assay (ELICA).

After incubation with marine extracts, the medium was withdrawncarefully using a pipetor and 100 μl of 10 mM Tris-HCl (pH 7.4) 150 mMNaCl, containing 3.7% formaldehyde was added to fix the cells for 30minutes at 4° C. The fixative was removed and replaced with 100 μl ofcold (−20° C.) methanol for 5 minutes to permeabilize the fixed cells.The methanol was removed and the wells were rinsed briefly with 200 μlantibody buffer. Then, 100 μl antibody buffer containing 0.1-0.15 μg/mlTG-3 monoclonal antibody and HRP-conjugated goat anti-mouse IgMsecondary antibody at a 50-fold dilution, was added to 16-20 hours at 4°C. The plates were washed twice with 200 μl 10 mM Tris-HCl pH 7.4, 0.02%Tween 20™. Then, 100 μl of 120 mM Na₂HPO₄, 100 mM citric acid (pH 4.0)containing 0.5 μg/ml 2,2′-azino-bis(-3ethylbenz-thiazoline-6-sulfonicacid) and 0.01% hydrogen peroxide was added for 1 hour at roomtemperature and the absorbance at 405 nm was measured.

Screens for Antimitotic Agents. MCF-7 cells were incubated for 20 hourswith different concentrations of the antimitotic drug paclitaxel, andthe proportion of cells arrested in mitosis was measured by countingmitotic cells in the microscope, and by ELISA. Paclitaxel inducedmitotic arrest in a concentration-dependent manner with half-maximalactivity at 10 nM measured by microscopy (FIG. 1A) and at 4 nM measuredby ELISA (FIG. 1B, □)

Dose-dependent arrest of cells in mitosis by paclitaxel was detected byELICA with half-maximal activity at 1.5 nM (FIG. 1B, (▪). ELICA provideda higher signal at low paclitaxel concentrations and a lower signal athigh concentrations as compared to ELISA. The differences may resultfrom higher non-specific staining of interphase cells because of reducedwashing and from lower specific staining of mitotic cells because offixation and reduced antibody incubation times. ELICA consistentlyshowed a difference in absorbance of 1 unit between cells treated or notwith antimitotic agents at concentrations causing maximal mitoticarrest, allowing unambiguous detection of mitotic cells. Measurementsobtained by ELICA consistently showed smaller standard deviations thanobtained by ELISA, because the reduced number of manipulations reducedexperimental variation. Thus, ELLCA is particularly suited for rapidscreening of large numbers of extracts while the ELISA assay may beuseful for more precise quantitation of antimitotic activity.

Screening of Biological Samples. EUISA was used to first screen a smallselection of crude extracts from marine microorganisms. Of 264 extractstested, 261 showed no activity, giving absorbance readings notstatistically different from those of untreated cells (0.270±0.051).Three extracts showed strong activity, with absorbance readings of1.135, 1.437 and 1.245, close to the values obtained with nocodazole asa positive control.

Over 2000 crude extracts of marine sponges, tunicates, gorgonians,starfish, and nudibianchs were then screened, initially by ELISA andlater by ELICA. This screen identified 16 additional extracts withantimitotic activity. The positive extracts were retested by countingmitotic figures in the microscope and all were found to arrest cells inmitosis.

Identification of Rhizoxin Analogs. Marine bacterial isolate MK7020collected off the coast of British Columbia, was identified as aPseudomonas sp. by gas chromatographic analysis of cellular fatty acids.Two active compounds (A and B described below) were purified bychrornatographic procedures using the ELISA to guide fractionation. Thetwo other microbial extracts were found to be independent isolates ofthe same Pseudomonas species and contained the same active compounds asMK7020. Compound A was identical to WF-1360, a previously reportedanalog of the antimitotic agent rhizoxin (Kiyoto, S. et al. (1986) 1.Antibiol. (Tokyo) 39:762-772; and, Iwaski, S. (1986) Chem. Pharm. Bull.34:1387-1390). Compound A showed half-maximal antimitotic activity(IC₅₀) at 52 nM as determined by ELISA. Compound B is a δ-lactone secohydroxy acid analog of rhizoxin, not previously known to be naturallyoccurring and which had an IC₅₀ of 8 nM.

Identification of New Antimitotic Terpenoids. An extra of octocoralEryihropodium caribaeorum collected from shallow reefs near Dominicaalso showed antimitotic activity. Eight active compounds were isolatedand their chemical structures elucidated, as described below.

Freshly collected specimens of E. caribaeorum were frozen on site andtransported to Vancouver over dry ice. Thawed samples (5.3 kg wet wt.)were extracted multiple times with MeOH and the combined MeOH extractswere concentrated to a gum in vacuo Fractionation of the crude gum (280g) by sequential application of vacuum reversed phase flash (gradientelution: 80:20 H₂O/MeOH to MeOH in 10% increments), normal phase flash(gradient elution: EtOAc to 80:20 EtOAc/McOH in 2% increments), andnormal phase high performance liquid chromatographies (HPLC) (eluent:93:7 CH₂Cl₂/MeOH) gave pure samples of 1 (50 mg), 2 (7 mg), 3 (6 mg), 4(3 mg), and 5 (2 mg). Compounds 6 (1 mg) and 7 (1 mg) partiallydecomposed on silica gel and were isolated using only vacuum reversedphase flash chromatography and cyano bonded phase HPLC (eluent: 56:42:2EtOAc/bexane/(iPr)₂NH). FIGS. 3A and 3B show the sequence of proceduresused to isolate eleutherobin, its analogs and other compounds from E.caribaeorum.

A major active compound was identified as being the known compoundeleutherobin 1. Compounds 2-7 described above were also identified.Desmethyleleutherobin 2 differs from eleutherobin by the presence of ahydroxyl instead of a methoxyl at C-4. Desacetyleleutherobin 3 retainsthe arabinose, but not the 2″ acetyl substituent of eleutherobin.Isoeleutherobin A 4 has an acetyl group at the 3″ position instead ofthe 2″ position. Z-eleutherobin 5 is the geometric isomer ofeleutherobin at the C-2′ to C-3′ double bond of the C-8N-(6)′-methylurocanic acid ester side chain. Caribacoside 6 differs fromeleutherobin by the addition of a hydroxy at C-11 of the tricyclic core,and a double bond at C-12 to C-13 instead of C-11 to C-12, therebyaltering the cyclohexene ring. Caribaeolin 7 differs from caribaeosideby the presence of a —CH₂OCO—CH₃ substituent in the C-3 side chain. Onefurther active compound was recovered and identified as the knowncompound, sarcodictyin A which differs from eleutherobin by replacementof the C-15 β-linked 2″-O-acetyl-D arabinopyranose side chain ofeleutherobin with a methyl ester and replacement of the C-4 methoxylwith a hydroxyl group.

The antimitotic activity profile of the above-described compounds asdetermined by ELICA is shown in FIG. 2. These results indicate an IC₅₀for eleutherobin of about 100 nM. The apparent IC₅₀ of Z-eleutherobinwas about 250 nM. Desmethyleleutherobin and isoeleutherobin A wereconsiderably more potent than eleutherobin, with an IC₅₀ of about 20 nMand about 50 nM, respectively. Desacetyleleutherobin was less potent,with an IC₅₀ of about 400 nM. Sarcodictyin A showed lower activity, withan IC₅₀ of about 2 μM. Caribaeoside and caribaeolin were considerablyless potent, with an IC₅₀ of about 20 μM for both compounds.

Further comparison of the antimitotic activities of eleutherobin anddesmethyleleutherobin was done using microscopic examination of astandard cell spread. The results shown in FIG. 4 indicate an IC₅₀ ofabout 25 nM for desmethyleleutherobin as compared to about 200 nM foreleutherobin.

Using the fractionation and assay procedures described above, similarantimitotic extracts were obtained from various other species from theorder Gorgonacea as well as species from the order Alcyoniidae.

In Vivo Antimitotic/Anticancer Activity. C3H mice of approximately 26 gbearing SCCVII squamous cell carcinoma (tumor size approximately 0.45 g)were injected intraperitoneally with desmethyleleutherobin. 24 hourslater, the mice were intravenously injected with Hoechst 33342™ and thensacrificed. The tumors were removed and single cell suspensions preparedand sorted on the basis of a concentration gradient of Hoechst 33342™,to provide cells sorted according to varying distance from the bloodsupply. The cells were plated and survival analyzed by observing colonyformation. The results are shown in FIG. 5. There was no overt toxicityto the mice and about 90% of the tumor cells were killed with doses ofabout 100 μg or more per mouse.

Characterization of Antimitotic Compounds. All NMR data for the E.caribaeorum diterpenes was recorded in DMSO-d₆ at 500 MHz. Eleutherobin1 was identified by comparison of its spectroscopic data with the valuesreported by Lindel, T. et al. [supr]. The UV chromophore foreleutherobin is: UV (MeOH) λ_(max) (log ε)−29 nm (3.8). Eleutherobincrystals were obtained which decomposed at 258-260° C.

Desmethyleleutherobin 2 was isolated as a clear oil that gave a[M+H]⁺ion in the HRFABMS at m/z 643.32230 appropriate for a molecularformula of C₃₄H₄₆N₂O₁₀ (ΔM−1.21 ppm), that differed from the molecularformula of eleutherobin by the loss of CH₂. The ¹H NMR spectrum of 2differed from the ¹H NMR spectrum of eleutherobin 1 only by the absenceof a methyl resonance at ≈δ 3.10 that could be assigned to the C-4methoxy substituent. 2D NMR data obtained for 2 was in agreement with anassignment of a hydroxyl group at C-4.

Desacetyleleutherobin 3 was isolated as a clear oil that gave a[M+H]⁺ion at m/z 615.32813 in the HRFABMS corresponding to a molecularformula of C₃₃H₄₆N₂O. (ΔM−0.05 ppm), that differed from the formula ofeleutherobin by the loss of C₂H₂O. The ¹H NMR spectrum of 3 showed astrong resemblance to the ¹H NMR spectrum of eleutherobin except for theabsence of a methyl singlet at ≈2 ppm that could be assigned to anacetyl residue and the chemical shifts of the resonances assigned to thearabinose protons. Acetylation of the abrabinose fragment of 3 withacetic anhydride in pyridine converted it to triacetyleleutlierobin,which was identical to triacetyleleutherobin prepared by acetylation ofeleutherobin using the same reaction conditions. Preparation oftriacetyleleutherobin by acetylation of eleutherobin was described in WO96/14745.

Isoeleutherobin A 4, isolated as a clear oil, gave a [M+H]⁺ ion at m/z657.33834 in the HRFABMS corresponding to a molecular formula ofC₃₅H₄₈N₂O₁₀ (ΔM−0.58 ppm), which was identical to the molecular formulaof eleutherobin. Comparison of the ¹H 1D and 2D NMR data forisoeleutherobin A 4 with the data for eleutherobin showed that themolecules differed only in the position of acetylation on the arabinosefragment. COSY correlations observed between resonances at δ 3.38 and3.62 (both broad doublets: J=11.5 Hz), assigned to the C-5″ methyleneprotons, and a methie at δ 3.83 (H4″: m) showed that the acetate was nota C-4″. The H-4″ resonance in turn showed a COSY correlation to aresonance at δ 4.80 (dd, J=10.1, 2.5 Hz), assigned to H3″, which wassignificantly deshielded relative to the corresponding H3″ resonance (δ3.73) in eleutherobin 1. Therefore, isoeleutherobin A was assignedstructure 4. Acetylation with acetic anhydride in pyridine convertedisoeleutherobin A 4 to diacetyleleutherobin 8, confirming the assignedstrcture of 4.

Z-Eleutherobin 5 gave a [M+H]⁺ ion at m/z 657.33830 in the HRFABMSappropriate for a molecular formula of C₃₅H₄₈N₂O₁₀ (ΔM−0.65 ppm), againidentical to the molecular formula of eleutherobin. Comparison of theNMR data obtained for 5 with the data for eleutherobin showed that themolecules differed only in the configuration of the Δ^(2′3′) olefin. Inthe ¹H NMR spectrum of Z-eleutherobin 5, the uroconic acid olefinicproton resonances appeared at δ 5.95 (H-2′) and 6.94 (H-3′) with acoupling constant of 12.6 Hz, whereas in the spectrum of eleutherobin,they were found at δ 6.35 (H-2′) and 7.35 (H-3′) with a couplingconstant of 15.6 Hz. The NMR sample of Z-eleutherobin 5 partiallyisomerized over time to eleutherobin, confirming the assigned structure.

Caribaeoside 6, obtained as a colorless glass, gave a [M+H]⁺ ion in theHRFABMS at m/z 673.33474 appropriate for a molecular formula ofC₃₅H₄₈N₂O₁₁ (ΔM−1.64 ppm), that only differed from the molecular formulaof eleutherobin 1 by the presence of one additional oxygen atom.Analysis of NMR data obtained for caribaeoside 6 revealed that it was aditerpene glycoside with the same N-(6′)-methylurocanic acid and2″-O-acetylarabinose substituents that are attached to the central coreof eleutherobin. A number of features of NMR data revealed thatcaribaeoside and eleutherobin differed in the C-11 to C-13 regions oftheir diterpene cores. The C-17 olefinic methyl resonance at δ 1.47 andthe H-12 olefinic methine resonance at δ 5.27 in the ¹H NMR spectrum ofeleutherobin (DMSO-^(d6)) were both missing in the ¹H NMR spectrum ofcaribaeoside 6. In their place, the ¹H NMR spectrum of 6 had a singletmethyl resonance at δ 0.82 and a pair of coincidentally chemical shiftequivalent olefinic methine resonances at δ 5.52 (H-12 and H-13). Thetwo proton olefinic resonance at δ 5.52 showed correlations in the HMQCspectrum to carbon resonance at δ 125.6 (C-13) and 137.5 (C-12). HMBCcorrelations observed between the Me-17 singlet at δ 0.82 and the C-12olefinic resonance at δ 137.5, a quaternary carbon resonance at δ 68.5,and a methine resonance at δ 45.8 (HMQC to δ 2.06) confirmed theproximity of Me-17 and C-12 and indicated that there was a hydroxylsubstituent at C-11 and a methine carbon at C-10. A pair of overlappingdoublet (6H) at δ 0.93-0.95, that showed COSY correlations to a methineresonance at δ 1.68, were assigned to the Me-19 and Me-20 isopropylprotons, and a multiplet at δ 4.00, that showed COSY correlations to anolefinic doublet at δ 5.38 (H-2) and a methine resonance at δ 2.06(H-10), was assigned to H-1. The H-1 resonance in the spectrum of 6 hada chemical shift and multiplicity nearly identical to the H-1 resonancein eleutberobin (δ 3,88), consistent with the proposal that the C-1,C-2, C-10, and C-14 centers in 6 were identical to the correspondingsites in 1. ROESY and scalar coupling constant data established therelative stereochemistry about the cyclohexene ring in caribaeoside 6.The resonances assigned to H-1 (δ 4.00) and H-2 (δ 5.38) in 6 hadchemical shifts and a vicinal coupling constant (J+9.7 Hz) nearlyidentical with their counterparts in eleutherobin (δ H-1, 3.88; H-2,5.39: J=9.4 Hz), indicating that the dihedral angle between them in 6was essentially identical to that in 1. ROESY correlations observedbetween the isopropyl methyl proton resonances at δ 0.93-0.95 and theH-1 (δ 4.00) and H-10 (δ 2.06) resonances in 6, demonstrated that theisopropyl group, H-1, and H-10 are on the same face of the molecule, asin cleutherobin. The Me-17 resonance at δ 8.02 in 6 showed a strongROESY correlation to the H-2 (δ 5.38) resonance demonstrating that Me-17and C-2 are cis. Models indicate that the Me-17 protons can sit in theshielding region of the Δ^(2,3) olefin, consistent with their unusuallyshielded chemical shift of δ 0.82.

Caribaeolin 7 was isolated as a clear oil that gave a [M+H]⁺ ion in theHRFARMS at m/z 541.29111 corresponding to a molecular formula ofC₃₀H₄₀N₂O₇ (ΔM−0.49 ppm). Analysis of the 1D and 2D ¹H detected NMR dataobtained for 7 showed that it contained the diterpene core andN-(6′)-methylurocanic acid fragments that constitute the aglycon ofcaribaeoside 6, but was missing the arabinose sugar residue. COSY andROESY correlations were observed between an olefinic methine resonanceat δ 5.37, assigned to H-2, and a broad two proton singlet at δ 4.46,assigned to the H-15 methylene protons. HMBC correlations were observedbetween a carbonyl resonance at δ 169.8 and both the H-15 methyleneproton resonance at δ 4.46 and a singlet methyl resonance at δ 1.97.These HMBC correlations demonstrated that in caribaeolin, a C-15 acetylsubstituent was present in place of the C-15 arabinose sugar residuefound in caribaeoside. Strong ROESY correlations were observed betweenthe Me-17 resonance at δ 0.77 and the H-2 olefinic proton resonance at δ5.37 indicating that Me-17 and C-2 were cis to each other as incaribaeoside 6, again accounting for the unusually shielded nature ofthe Me-17 proton resonance. Additional ROSEY correlations observedbetween the C-19/C-20 isopropyl methyl proton resonance at δ 0.94-0.95and the H-1 (δ 4.01) and H-10 (δ 2.08) resonances confirmed that theisopropyl group, H-1 and H-10 were all on the same face of the molecule.

The significant decrease in antimitotic potency of caribaeoside 6relative to eleutherobin 1, resulting from introduction of a hydroxylgroup at C-11 and migration of the olefin to the Δ^(12,13) position,alters both the shape and polarity of region B of the proposedpharnacophore. The Ojima pharmacophore proposal suggests that changes inthe C-11 to C-13 region of eleutherobin would have an impact on theability of analogs to stabilize tubulinpolyrers.

Replacement of the arabinose fragment in caribaeoside 6 with a simpleacetate reside (Compound 7) results in no additional loss of potency.Altering the Δ^(2′,3′) configuration (a change in the A region of thepharnacophore) has little effect (e.g. Compound 5), while alterations inplacement of an acetyl group on the arabinose fragment (representingchanges in the C region of the pharmacophore) can either enhance (e.g.Compound 4) or decrease potency (e.g. Compound 3). Changing the C-4substituent from the methoxyl of eleutherobin to hydroxyl or a C₂-C₁₀alkyl as described below, which are alterations that are outside of theOjima pharnacophore binding regions are now shown to result in anincrease in potency.

Eleutherobin is an Artifact Derived From Desmethyleleutherobin.Erythropodium caribaeorum was exhaustively extracted with ethanol atroom temperature and the ethanol extract was fractionated as describedabove (and in FIG. 3) for the methanol extract. This procedure yieldedonly desmethyleleutherobin and the ethylketal analog of eleutherobinwhich has a C-4 ethoxyl group. Eleutherobin was not detected. Thisdemonstrates that the methyl ketal of C-4 in eleutherobin is an artifactformed by reaction of desmethyleleutherobin with a methanol extractionsolvent in the first instance. We have found that desmethyleleutherobincan be converted quantitatively to eleutherobin by treatment with acatalytic amount of pyridinium p-toluenesulfonate in methanol at roomtemperature. Simply leaving desmethyleleutherobin sitting in methanolwithout pyridinium p-toluenesulfonate results in no detectableconversion to eleutherobin. However, the initial crude methanol extractof E. caribaeorum has a pH of approximately 4, which is acidic enough tocatalyze the conversion of desmethyleleutherobin to eleutherobin duringthe extraction procedure with methanol or to the ethylketal analogduring an extraction procedure with ethanol. Therefore, a preferredextraction procedure for obtaining exclusively desmethyleleutherobininvolves freeze-drying freshly collected samples (to reduce watercontent) and then extracting pulverized dried material multiple timeswith ethyl acetate. Concentration of combined ethyl acetate extracts invacuo provides a crude extract that could be purified as described aboveto give pure desmethyleleutherobin without the formation ofeleutherobin.

Antimitotic Ketal Analogs. Desmethyleleutherobin was reacted with(independently) ethanol, propanol and n-butanol in the presence ofpyridinium p-toluenesulfanate to produce the respective ketal forms atC-4. Surprisingly, these analogs demonstrated greater potency in theELICA assay than eleutherobin. The approximate IC₅₀ value obtained was30 nM for the ethoxyl, propoxyl, and butoxyl forms.

All publications, patents and patent applications referred to herein arehereby incorporated by reference. While this invention has beendescribed according to particular embodiments and by reference tocertain examples, it will be apparent to those of skill in the art thatvariations and modifications of the invention as described herein fallwithin the spirit and scope of the attached claims.

We claim:
 1. A method to obtain an antimitotic compound wherein ahomogenate of one or more organisms of the order Gorgonacea in a solventis fractionated to separate an antimitotic terpenoid compound fromcompounds lacking antimitotic activity that are present in thehomogenate, wherein the organism is a gorgonian coral.
 2. The method ofclaim 1 wherein the solvent is suitable to extract a terpenoid compoundfrom the organism.
 3. The method of claim 2 wherein the solvent is analcohol.
 4. The method of claim 2 wherein the solvent is ethyl acetate.5. The method of any one of claims 1-4 wherein the homogenate isfractionated by chromatography.
 6. The method of any one of claims 1-4wherein the presence of the antimitotic terpenoid compound is detectedby an assay for antimitotic activity.
 7. The method of any one of claims1-4 wherein the organism is of the genus Erythropodium.
 8. The method ofclaim 7 wherein the organism is E. caribaeorum.
 9. The method of any oneof claims 1-4 wherein the compound is selected from the group consistingof desmethyleleutherobin, eleutherobin, isoeleutherobin A,Z-eleutherobin, desacetyleleutherobin, caribaeoside, caribaeolin andsarcodictyin A.